Prophylactic agent for heart failure

ABSTRACT

The present invention provides an agent having a suppressive action against the thickening of the heart wall, a prophylactic agent for heart failure containing the above described agent, and functional foods expected to have a prophylactic effect for heart failure. The agent for suppressing heart wall thickening comprises Xaa Pro Pro as an active ingredient.

FIELD OF THE INVENTION

The present invention relates to an active ingredient having asuppressive action on cardiac wall thickening, a prophylactic agent forcardiac hypertrophy and a prophylactic agent for heart failurecontaining the active ingredient, as well as functional foods containingthe active ingredient, the functional foods being expected to have aprophylactic effect against cardiac hypertrophy and heart failure.

Also, the present invention relates to a method for suppressing cardiacwall thickening. Further, the present invention relates to a method forpreventing cardiac hypertrophy and heart failure.

BACKGROUND OF THE INVENTION

Recently, the number of those who have a risk factor considered to causeheart diseases, such as smoking, hypertension, hyperglycemia, andhyperlipidemia, have increased in some countries. There is an increasingtrend in the number of patients suffering from heart failure. Inaddition, these pathologic states are not limited to human and theincreasing trend in morbidity of such diseases are seen among animalsthat closely interact with human, such as dogs, cats, other companionanimals and pet animals.

Since the pathologic state, classification, and progression of heartfailure varies, the disease cannot be readily defined. Yet it can beroughly divided into two types: systolic heart failure and diastolicheart failure. In some cases, these two types simultaneously occur.Systolic heart failure occurs when the heart fails to contract normally.The heart can take in blood but cannot fully pump out adequate blood dueto weakened cardiac muscles. As a result, the volume of the bloodpumping out to the whole body and lungs decreases and the heart, inparticular the left ventricle, can become hypertrophic. On the otherhand, diastolic heart failure occurs when the heart wall becomes toostiff to fill up the heart with blood. As a result, blood dams up in theleft atrium and lung blood vessels, which could cause congestion. Thus,heart failure generally refers to a condition that heart's pumpfunctions become impaired and hence the heart become incapable ofpumping a sufficient amount of blood. Heart failure could cause otherchanges which further deteriorate functions of the heart, including adecrease in blood volume and congestion of blood in the veins and lungs.

Examples of primary diseases that may cause heart failure includeincreased pressure overload by hypertension, aortostenosis and the like,cardiac hypertrophy with heart wall thickening by volume overload andthe like by valvular disease. Meanwhile, after the onset of myocardialinfarction, heart wall thickening may be developed in injury tissues asa spontaneous reaction for functional recovery. Also, agnogenicmyocardiopathy of unexplained origins, such as myocardosis, may alsocause heart wall hypertrophy. Continuous mechanical load to the hearttriggers a gradual decrease in the contractile power of the cardiacmuscles, followed by reduction in cardiac performance, and eventuallyprogresses to ventricular arrhythmia, heart ischemia, coronary arterydisease, and congestive heart failure.

Considering the situation, a compound having the suppressive actionagainst heart wall thickening is useful for prophylaxis and treatment ofcardiac hypertrophy and heart failure.

As a method for improving heart wall thickening, in the case of cardiachypertrophy caused by hypertension and the like, for instance, treatingstem-cause hypertension can improve heart wall thickening in some cases.However, the action is not satisfactory and also some antihypertensivedrugs have no effects (Am J. Hypertens. 1997 August; 10(8):913˜20).Also, even when hypertension is improved, heart wall thickening remainsunimproved in many cases. In such cases there is still a risk for theonset of heart failure.

Effective drugs for treating heart failure include angiotensinconverting enzyme inhibitors (ACEI), such as Enalapril, which inhibitsan enzyme converting angiotensin I to angiotensin II having ahypertensive action (i.e. angiotensin converting enzyme; ACE), and has ahypotensive action. In addition, the antihypertensive drug reportedlyimproves the progression of renal damage at the same time as loweringblood pressure (J. Clin. Invest., 77, 1993-2000, 1986). Yet on the otherhand, for heart failure induced by various primary diseases, the ACEinhibitors are not effective for all cases. Rather there is a risk ofdeveloping acute renal failure accompanying lowering blood pressure andtherefore it has been pointed out that careful administration isrequired (Saishin Igaku, 48:1404 to 1409, 1993), That is, theantihypertensive drug can be insufficient for the prophylaxis of heartfailure but also could rather induce renal failure.

Meanwhile, it has been reported that a peptide originated from foodmaterials, such as casein has the ACE inhibitory activity and suchpeptides are known to have a hypotensive action. However, whether theyhave a suppressive action against heart wall thickening or not has notbeen directly demonstrated (Japanese Patent Publication No. 2782142, J.Dairy Sci. 1995, 78:777-78, J. Dairy Sci. 1995, 78:1253-1257, Am. J.Clin. Nutr. 1996, 64:767-771). Additionally, as described above, sincethe prophylactic and therapeutic effect for heart failure mediated byACE inhibition is limited, prophylaxis and treatment of the diseasewhich are not dependent on the ACE inhibition are desired.

SUMMARY OF THE INVENTION

The present invention provides a compound or composition having asuppressive action against heart wall thickening independently of theACE inhibitory activity.

Further the present invention provides a prophylactic agent for heartfailure containing the above described compound or composition.

In addition, the present invention provides functional foods expected tohave a prophylactic effect for heart failure containing the abovedescribed compound or composition.

Further the present invention provides a method for suppressing heartwall thickening, which method includes administration of the abovedescribed compound or composition to a subject.

In addition, the present invention provides a method for preventingheart failure, which method includes administration of the abovedescribed compound or composition to a subject.

The present invention is also a use of the above described compound orcomposition in production of a pharmaceutical for suppressing heart wallthickening. In particular, the present invention is a use of the abovedescribed compound or composition in production of a pharmaceutical forpreventing cardiac hypertrophy. Yet, the present invention is a use ofthe above described compound or composition in producing apharmaceutical for preventing heart failure.

The present inventors have discovered that a tripeptide having aspecific structure Xaa Pro Pro (wherein Xaa is any naturally occurringamino acid) has a suppressive action against heart wall thickening, andhave reached to invent a pharmaceutical and functional foods useful forprophylaxis of heart failure.

Concrete Contents of the Present Invention are as Follows:

The present invention provides an agent for suppressing heart wallthickening containing Xaa Pro Pro as an active ingredient.

The present invention also provides a prophylactic agent for cardiachypertrophy having the above described suppressive action against heartwall thickening.

In addition, the present invention provides a prophylactic agent forheart failure having the above described suppressive action againstheart wall thickening.

In addition, the present invention provides functional foods containinga prophylactic agent for heart wall thickening.

The present invention provides a method for suppressing heart wallthickening, which method includes administration of Xaa Pro Pro or acomposition containing Xaa Pro Pro to a subject.

In addition, the present invention also provides a method for preventingcardiac hypertrophy, which method includes administration of Xaa Pro Proor a composition containing Xaa Pro Pro to a subject.

In addition, the present invention provides a method for preventingheart failure, which method includes administration of Xaa Pro Pro or acomposition containing Xaa Pro Pro to a subject.

The present invention is also a use of Xaa Pro Pro or a compositioncontaining Xaa Pro Pro in producing a pharmaceutical for suppressingheart wall thickening. In particular, the present invention is also ause of Xaa Pro Pro or a composition containing Xaa Pro Pro in producinga pharmaceutical for preventing cardiac hypertrophy. The presentinvention is also a use of Xaa Pro Pro or a composition containing XaaPro Pro in producing a pharmaceutical for preventing heart failure.

Preferably Xaa Pro Pro is Val Pro Pro and/or Ile Pro Pro.

In other embodiment of the present invention, the active ingredient XaaPro Pro is derived from an animal milk casein hydrolysate or aconcentrate thereof.

In other further embodiment of the present invention, the activeingredient Xaa Pro Pro is originated from a fermented product obtainedby fermenting a raw material containing a milk protein with a bacteriumbelonging to the species Lactobacillus helveticus.

Preferably the bacterium belonging to the species Lactobacillushelveticus is Lactobacillus helveticus CM4 strain (FERM BP-6060).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of Test 1 examining the effect of Val Pro Pro(VPP) to suppress rat left ventricle wall thickening. The test wasconducted using the group of rats administered with L-NAME (11 rats pergroup), the group of rats administered with L-NAME and Enalapril (9 ratsper group), and the group of rats administered with L-NAME and VPP (12rats per group). The graph represents mean±standard error. Comparisonswere demonstrated by the one-sided t-test. The symbol (*) indicatesstatistical significance (p<0.05).

FIG. 2 shows the results of Test 2 examining the effect of Val Pro Pro(VPP) to suppress rat right ventricle wall thickening. The test wasconducted using the group of rats administered with L-NAME (11 rats pergroup), the group of rats administrated with L-NAME and Enalapril (9rats per group), and the group of rats administered with L-NAME and VPP(12 rats per group). The graph represents mean±standard error.Comparisons were demonstrated by the one-sided t-test. The symbol (*)indicates statistical significance (p<0.05).

FIG. 3 shows the results of Test 3 examining the effect of Ile Pro Pro(IPP) to suppress rat left ventricle wall thickening. The test wasconducted using the group of rats administered with L-NAME (9 rats pergroup), the group of rats administered with L-NAME and Enalapril (9 ratsper group), and the group of rats administered with L-NAME and IPP (11rats per group). The graph represents mean±standard error. Comparisonswere demonstrated by the one-sided t-test. The symbol (#) indicatesstatistical significance (p<0.1).

FIG. 4 shows the results of Test 5 examining the effect of Ile Pro Pro(IPP) to suppress rat heart septal wall thickening. The test wasconducted using the group of rats administrated with L-NAME (9 rats pergroup), the group of rats administered with L-NAME and Enalapril (9 ratsper group), and the group of rats administered with L-NAME and IPP (11rats per group). The graph represents mean±standard error. Comparisonswere demonstrated by the one-sided t-test. The symbol (*) indicatessignificant difference (p<0.05).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The agent for suppressing heart wall thickening according to the presentinvention comprises a tripeptide with a specific structure of Xaa ProPro as an active ingredient. The term “agent” herein is not restrictedto a pharmaceutical but refers to a composition such as a pharmaceuticalcomposition or food composition, or a compound such as a food additive.The term “agent for suppressing heart wall thickening” used in thepresent invention refers to a compound or composition having an actionto suppress progress of lesions of heart wall thickening, an action toimprove lesions of heart wall thickening, or a prophylactic effect forthe onset of heart wall thickening.

The Xaa in the active ingredient Xaa Pro Pro according to the presentinvention may be any naturally occurring amino acid. Concrete examplesinclude Val Pro Pro (valine proline proline), Ile Pro Pro (isoleucineproline proline), Ser Pro Pro (serine proline proline), and Leu Pro Pro(leucine proline proline). Preferred are Val Pro Pro and Ile Pro Prowith Val Pro Pro being more preferred. As the active ingredient, theagent may contain a combination of two or more of the tripeptides XaaPro Pro, as exemplified by a combination of Val Pro Pro and Ile Pro Pro.

The active ingredient Xaa Pro Pro may be an chemically synthesizedtripeptide or naturally occurring tripeptide.

As an chemical synthetic method of tripeptide Xaa Pro Pro, a commonmethod such as solid phase method (Boc method and Fmoc method) andliquid phase method can be employed. For instance, the peptide may besynthesized using an automatic peptide synthesizer such as a peptidesynthesizer (PSSM-8type) manufactured by Shimadzu Corporation. Withregard to reaction conditions for the peptide synthesis and the like,those skilled in the art can arbitrarily set appropriate reactionconditions and the like, based on their technological common knowledgedepending on the synthesis method to be selected and desired tripeptideXaa Pro Pro.

Alternatively, as the naturally occurring peptide, the tripeptide may beoriginated from an animal milk casein hydrolysate or a concentratethereof, as well as may be originated from a fermented product obtainedby fermenting a food material containing a protein with fungi orbacteria such as Aspergillus and Lactobacillus.

In cases where the animal milk casein hydrolysate or the concentratethereof, or the fermented product obtained by fermenting the rawmaterial containing the milk protein with the bacterium belonging to thespecies Lactobacillus helveticus, besides the tripeptide Xaa Pro Prowhich is the active ingredient according to the present invention, freeamino acids may be contained. Furthermore, in addition to the abovedescribed peptide and free amino acids, for example, lipids, ash,carbohydrates, dietary fibers, water and the like, all of which arenormally contained in commercially available animal milk caseins or milkproteins may be contained. Additionally, as required, a part or all ofthe appropriate components among these may be taken out.

The active ingredient Xaa Pro Pro according to the present invention maybe originated from an animal milk casein hydrolysate or a concentratethereof obtained by a method of hydrolyzing an animal milk casein with agroup of enzymes yielding Xaa Pro Pro, in particular Val Pro Pro and IlePro Pro and/or a method of fermenting animal milk with Aspergillus.

Examples of the animal milk casein include cow milk, horse milk, goatmilk, and ewe milk. In particular, cow milk casein being preferablyused.

A concentration of casein when the animal milk casein is hydrolyzed orfermented is not restricted but is preferably 1 to 19% by weight inorder to efficiently produce the animal milk casein degraded product.

An example of enzyme group is preferably an enzyme group (X) including apeptidase capable of cleaving between Pro and Xaa residues at thecarboxyl terminus of the Xaa Pro Pro Xaa sequence.

The enzyme group (X) is preferably a serine type proteinase havingserine in the active center or a metal proteinase having a metal in theactive center. Examples of the metal proteinase include neutral proteaseI, neutral protease II and leucyl aminopeptidase. It is preferred thatat least one type of these metal proteinases be additionally included inthat the desired hydrolysates can be efficiently obtained in a shorttime, and even in a one-step reaction. Additionally the peptidasecapable of cleaving the above described Pro Xaa sequence is preferablyan enzyme showing the isoelectric point in the acidic region.

An example of the above described enzyme group or enzyme group (X) is anenzyme group originated from Aspergillus such as Aspergillus oryzae.Such an enzyme group includes an enzyme group which fungi cells werecultured in an appropriate culture medium and an enzyme produced wasextracted with water. Among the enzyme groups originated fromAspergillus oryzae, an enzyme group showing the isoelectric point in theacidic region is in particular preferred.

As the enzyme group originated from Aspergillus oryzae, commerciallyavailable products can be used. Examples thereof are Sumizyme FP, LP orMP(all registered trademark, manufactured by SHINNIHON CHEMICALSCorporation), Umamizyme (registered trademark, manufactured by AmanoEnzyme Inc.), Sternzyme B11024, PROHIDROXY AMPL(all trade names,manufactured by Higuchi Shoukai Co., Ltd.), Orientase ONS (registeredtrademark, manufactured by Hankyu Bioindustry CO., LTD), Denatyme AP(registered trademark, manufactured by Nagase ChemteX Corporation) withSumizyme FP(registered trademark, SHINNIHON CHEMICALS Corporation) beingpreferably used.

When these commercially available enzyme groups are used, optimumconditions are usually pre-determined. Yet, conditions such as an amountof enzyme to be used and reaction time can be appropriately altereddepending on an enzyme group to be used such that the above describedcasein hydrolysate can be obtained.

An amount of the enzyme group to be added when the above describedanimal milk casein is hydrolyzed is, for instance, such that the weightratio of enzyme group/animal milk casein in a aqueous solution in whichthe animal milk casein is dissolved is not less than 1/1000, preferably1/1000 to 1/10, especially preferably 1/100 to 1/10, further preferably1/40 to 1/10.

The reaction conditions can be appropriately selected depending on anenzyme group to be used such that an intended casein hydrolysate isobtained. A temperature is usually 25 to 60° C., preferably 45 to 55° C.And pH is 3 to 10, preferably 5 to 9, particularly preferably 5 to 8.The enzyme reaction time is usually 2 to 48 hours, preferably 7 to 15hours.

Termination of the enzyme reaction can be achieved by inactivating theenzyme. Usually the enzyme is inactivated at 60 to 110° C. to stop thereaction.

After the enzyme reaction is terminated, as required, it is preferred toremove precipitates by centrifugation removal and various filtertreatments.

Additionally, as required, a peptide having bitterness and/or smell canbe removed from the obtained hydrolysate. The removal of such abitterness component and/or smell component can be done using activatedcharcoals, hydrophobic resins or the like. For instance, the removal canbe carried out by adding the activated charcoals to the obtainedhydrolysate in 1 to 20% by weight based on an amount of casein used andby allowing to react the resulting mixture for 1 to 10 hours. Removal ofthe activated charcoals can be carried out by a known method such ascentrifugation and membrane treatment process.

The thus obtained reaction mixture containing the animal milk caseinhydrolysate or the concentrate thereof can be added as is to a liquidproduct such as beverage to be use in the functional foods. In order toimprove versatility of the animal milk casein hydrolysate, the abovedescribed reaction mixture can be, after concentrated, dried to yield apowdered form.

The content ratio of Xaa Pro Pro contained in the animal milk caseinhydrolysate or the concentrate thereof is usually not less than 1% byweight, preferably 1 to 5% by weight based on a total amount of peptidesand free amino acids in the animal milk casein hydrolysate or theconcentrate thereof. By having the content ratio of not less than 1% byweight, higher actions are expected. Additionally, either when thecontent ratio of each of Ile Pro Pro or Val Pro Pro contained in theanimal milk casein hydrolysate or the concentrate thereof is not lessthan 0.3% by weight based on a total amount of peptides and free aminoacids in the animal milk casein hydrolysate or the concentrate thereof,or when the content ration of a total of Ile Pro Pro and Val Pro Pro isnot less than 0.3% by weight, high effects are expected. Further, when0.3% by weight or more of each of Ile Pro Pro and Val Pro Pro iscontained, higher effects are expected.

The active ingredient Xaa Pro Pro according to the present invention maybe also originated from a fermented product obtained by fermenting a rawmaterial containing a milk protein with a bacterium belonging to thespecies Lactobacillus helveticus. Although the bacterium belonging tothe species Lactobacillus helveticus is preferably used individually inthe fermentation, other Lactobacillus or the like may beincluded/contained to the extent that the desired effects according tothe present invention is not adversely affected.

As the bacterium belonging to the species Lactobacillus helveticus, aproteinase-producing bacterium capable of highly producing Ile Pro Proand/or Val Pro Pro is preferred. For instance, a bacterium strainshowing a U/OD590 value measured in accordance with a method by Yamamotoet al. (Yamamoto, N. et al. J. Biochem.) (1993) 114, 740), which isbased on a method by Twining et al. (Twining, S. Anal. Biochem.) 1433410 (1984)), of not less than 400 is preferred.

An example of such a preferred bacterium strain is Lactobacillushelveticus CM4 strain (Ministry of International Trade and Industry,National Institute of Bioscience and Human-Technology, 1-1-3 Higashi,Tsukuba, Ibaraki, Japan, Zip code 305, (currently, International PatentOrganism Depositary, National Institute of Advanced Industrial Scienceand Technology, AIST, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,Ibaraki, Japan, Zip code 305-8566), Accession No.: FERM BP-6060, Date ofdeposit: Aug. 15, 1997) (hereinafter referred to as CM4 strain). CM4strain has been registered under the above described accession numberunder the Budapest Treaty concerning an international approval ofmicroorganism depository on patent procedures, and already patented.

The fermented product obtained by fermenting a raw material containingmilk protein with the bacterium belonging to the species Lactobacillushelveticus can be obtained by adding a fermented milk starter containingthe bacterium strain belonging to the species Lactobacillus helveticusto a raw material containing the milk protein and by fermenting themixture on appropriately selected conditions such as fermentationtemperature.

A concentrate of the thus obtained fermented product or the like may bepowdered with freeze drying, spray drying, or the like and may be usedas a powder.

The bacterium belonging to the species Lactobacillus helveticus ispreferably used as a starter with sufficiently high activities, which ispreliminarily pre-cultured. The number of the bacteria at the beginningis preferably about 10⁵ to 10⁹ cells/ml.

The fermented product obtained by fermenting a raw material containing amilk protein with the bacterium belonging to the species Lactobacillushelveticus, for example, when used for functional foods such as Foodsfor Specified Health Uses, can be also fermented with yeast inconjunction with the above described bacterium strain belonging to thespecies Lactobacillus helveticus in order to have better flavor andbetter palatability. An example of the strain of yeast is preferably thegenus Saccharomyces such as Saccharomyces cerevisiae but not restrictedthereto. The content ratio of the yeast can be appropriately selecteddepending on a purpose.

Examples of the raw material containing the milk protein include animalmilk such as cow milk, horse milk, ewe milk, goat milk, vegetable milksuch as soybean milk, and processed milk of these milk including skimmedmilk, reconstituted milk, powdered milk, and condensed milk. Cow milk,soybean milk and processed milk of these milk is preferred and cow milkor processed milk thereof is in particular preferred.

Although the solid content concentration of the milk is not particularlyrestricted, when the skimmed milk is for instance used, the solidcontent concentration of non-fat milk is usually about 3 to 15% byweight and preferably 6 to 15% by weight for better productivity.

The above described fermentation is usually carried out by standing orstirring fermentation, for example, by a method of fermenting at atemperature of 25 to 45° C., preferably 30 to 45° C., with afermentation time of 3 to 72 hours, preferably 12 to 36 hours, whereinthe fermentation is terminated when lactic acid acidity reaches 1.5% ormore.

The content ratio of Xaa Pro Pro, preferably Ile Pro Pro and/or Val ProPro in the fermented product obtained by fermenting the raw materialcontaining the milk protein with the bacterium belonging to the speciesLactobacillus helveticus is preferably 10 mg or more, preferably 15 mgor more, in terms of 100 g of a freeze dried product of the fermentedproduct.

As for a dose or intake per day of the agent for suppressing heart wallthickening according to the present invention, in the case of human,usually a dose of about 10 μg to 10 g. preferably 1 mg to 5 g, furtherpreferably 3 mg to 1 g of the active ingredient Xaa Pro Pro, preferablyVal Pro Pro and/or Ile Pro Pro may be administrated or taken dividedlyin several times in a day.

A period of administration or intake of the agent for suppressing heartwall thickening can be adjusted in many ways in view of age of humans oranimals to be administrated or to ingest and/or environments for a riskfactor for heart wall thickening of the humans or animals. The periodcan be for example usually one day or more, preferably 7 days to 365days.

The prophylactic agent for heart failure according to the presentinvention comprises the above described tripeptide as an activeingredient.

As for a dose or intake per day of the prophylactic agent for heartfailure according to the present invention, in the case of human,usually a dose of about 10 μg to 10 g. preferably 1 mg to 5 g, furtherpreferably 3 mg to 1 g of the active ingredient Xaa Pro Pro, preferablyVal Pro Pro and/or Ile Pro Pro may be administrated or taken dividedlyin several times in a day.

A period of administration or intake of the prophylactic agent for heartfailure can be adjusted in many ways in view of age of human or animalsto be administrated or ingest and/or environments for a risk factoragainst heart failure of the human or animals. The period can be forexample usually 1 day or more, preferably 7 days to 365 days.

The prophylactic agent for cardiac hypertrophy according to the presentinvention contains the above described tripeptide as an activeingredient.

A dose or intake and period of administration or intake of theprophylactic agent for cardiac hypertrophy according to the presentinvention are same as those of the prophylactic agent for heart failureaccording to the present invention.

A method of administration or intake of the agent for suppressing heartwall thickening, prophylactic agent for cardiac hypertrophy andprophylactic agent for heart failure according to the present inventionis preferably oral administration.

A form of the agent for suppressing heart wall thickening, prophylacticagent for cardiac hypertrophy, prophylactic agent for heart failureaccording to the present invention, when used as a pharmaceutical, is aform of formulation for oral administration. Examples thereof includetablet, pill, hard capsules, soft capsules, microcapsule, powder,pellet, and solution.

In the case of preparing as a pharmaceutical, for example,pharmaceutically acceptable carrier, adjuvant, vehicle, excipient,antiseptics, stabilizing agent, binder, pH adjusting agent, bufferagent, thickener, gelatinizer, preservatives, anti-oxidant or the likecan be, as required, used. The pharmaceutical can be produced in a unitdose form required pharmaceutical formulation administration generallyaccepted.

The foods according to the present invention comprises the agent forsuppressing heart wall thickening according to the present invention asan active ingredient, and can be, for example, functional foods, such asFoods for Specified Health Uses, which has efficacies such the actionfor suppressing heart wall thickening, prophylaxis for cardiachypertrophy, and prophylaxis for heart failure.

An intake to attain such efficacies, for example, in the light of thefact that the functional foods are daily, continuously, orintermittently taken for a long period of time, in the case of human, isusually about 10 μg to 10 g per day, in terms of an amount of the activeingredient Xaa Pro Pro or an amount of Val Pro Pro and/or Ile Pro Pro,preferably 1 mg to 5 g, more preferably 3 mg to 1 g. Depending on thenumber of intake per day, an intake of the foods such as the functionalfoods per once can be reduced further to a lower amount than the amountdescribed above.

In cases where the animal milk casein hydrolysate or the concentratecontaining the active ingredient Xaa Pro Pro is used as is, in the caseof human, the hydrolysate or the concentrate thereof is preferably takenusually in 1 mg to 100 g per day, particularly in about 100 mg to 10 g.

In cases where the freeze dried product of the fermented productcontaining the active ingredient Xaa Pro Pro is used as is, in the caseof human, usually 1 to 100 g per day in terms of a dried amount of thefreeze dried product of the fermented product, in particular, about 2 to50 g is preferably taken.

A period of the intake of the foods according to the present invention,for example the functional foods, is not particularly restricted. Theintake for a long period of time is preferred. In order to attain theabove described efficacies, for example, the period can be usually notless than one day, preferably 7 days to 365 days.

The foods according to the present invention, for example functionalfoods, comprise the agent for suppressing heart wall thickeningcontaining the active ingredient Xaa Pro Pro, preferably Val Pro Proand/or Ile Pro Pro. For instance, the animal milk casein hydrolysate orthe concentrate thereof and the fermented product, all of which wereobtained in the above described manner, can be added to a variety offoods as they are or in the form of powder or granule. If necessary, afermented product by Lactobacillus other than Lactobacillus helveticus,other ingredients used in foods including sugars, proteins, lipids,vitamins, minerals, or flavors, or additives such as variouscarbohydrates, lipids, vitamins, minerals, sweeteners, flavors, coloringagents, texture improving agents or a mixture thereof may be added toimprove nutritional balance, flavor, and/or the like.

The foods according to the present invention, for example the functionalfoods, can be any form of solids, gels, or liquids. Examples thereofinclude fermented dairy products such as lactobacillus beverages, avariety of processed foods and beverages, dried powders, tablets,capsules, and granule. Further, additional examples can be variousbeverage, yogurts, fluid diets, jellys, candies, retort-packed foods,tablet candies, cookies, Castella (or Japanese sponge cake), breads,biscuits, and chocolates.

The present invention will now be described in detail by way of examplesthereof, but the scope of the present invention is by no means limitedby the examples.

EXAMPLES Example of Peptide Synthesis

The active ingredient according to the present invention Ile Pro Pro andVal Pro Pro were synthesized by the following chemical synthesizingmethod (Fmoc method). The synthesis was carried out by solid phasemethod using an automatic peptide synthesizer (PSSM-8type) manufacturedby Shimadzu Corporation.

As a solid phase carrier, 50 mg of a resin (SynProPep Resin®,manufactured by Shimadzu Corporation) which is a2-chlorotrityl(2-Chlorotrityl)type polystyrene resin in which prolineprotected its amino group with a fluorenylmethyloxy carbonyl group(hereinafter referred to as Fmoc for short) is bound, was used.According to the above described amino acid sequence, Fmoc-Ile,Fmoc-Pro, and Fmoc-Val (100 μmol each) which are protected their aminogroup with the Fmoc group were reacted in the order of the peptidesequence to obtain a peptide-bound resin in accordance with aconventional method.

This peptide-bound resin was then suspended in 1 ml of a reactionmixture A (10 volume % acetic acid, 10 volume % trifluoroethanol, 80volume % dichloromethane), allowed to react at room temperature for 30to 60 minutes. Thereafter the peptides were separated from the resin andthe reaction mixture A was filtered with a glass filter. Immediatelyafter the solvent in the filtrate was removed under reduced pressure, 1ml of reaction mixture B (82.5 volume % trifluoroacetic acid, 3 volume %ethyl methyl sulfide, 5 volume % purified water, 5 volume % thioanisole,2.5 volume % ethanedithiol, 2 volume % thiophenol) was added and themixture was allowed to react at room temperature for six hours touncouple the side chain protecting group. To the resultant 10 ml ofanhydrous ether was added to precipitate the peptide, and centrifuged at3000 revolutions for five minutes to separate. The precipitate waswashed with anhydrous ether was several times and then dried by sprayingnitrogen gas. The entire volume of the thus obtained unpurifiedsynthetic peptide was dissolved in 2 ml of 0.1N hydrochloric acidaqueous solution and then HPLC using a C18 reverse phase column wascarried out in accordance with the conditions below.

Pump: Type L6200 intelligent pump (Hitachi, Ltd); detector: Type L4000UVdetector (Hitachi, Ltd) for detection of ultra-violet absorption at 215nm; column: Micro Bondashere5μC18 (Waters Corp.); eluting solutions:solution A; 0.1% by weight TFA aqueous solution, solution B;acetonitrile with 0.1% by weight TFA aqueous solution; (B/A+B)×100(%):0→40% (60 minutes); and flow rate: 1 ml/minute. The eluted fractionshowing the maximum absorbance was collected and then freeze-dried toobtain the intended synthetic peptide Ile Pro Pro and Val Pro Pro (5.7mg and 6.5 mg, respectively). The purified peptide was analyzed using anautomatic protein primary structure analyzer (Type PPSQ-10, manufacturedby Shimadzu Corporation) from the N-terminal of the peptide. Thepurified peptide was further analyzed an amino acid analyzer (Type 800series, manufactured by JASCO corporation) to confirm that the peptidewas the one as designed.

[Example of Preparation of Animal Milk Casein Hydrolysates]

To Casein originated from cow milk (Nippon NZMP Ltd.) (1 g) 99 g ofdistilled water adjusted to about 80° C. was added and the mixture wasstirred well. Subsequently, 1N sodium hydroxide (manufactured by WakoPure Chemical Industries, Ltd.) solution was added. The mixture was thenadjusted to pH 7.0 and a temperature of 20° C. to prepare a substratesolution.

To the obtained substrate solution a commercially available enzyme(registered trademark “Sumizyme FP”, manufactured by SHINNIHON CHEMICALSCorporation) was added, which was derived from Aspergillus oryzae andcontains at least metal protease, serine protease, neutral protease I,neutral protease II and leucyl aminopeptidase such that theenzyme/casein weight ratio of 1/25 was attained. The mixture was allowedto react at 50° C. for 14 hours. Subsequently, the enzymes wereinactivated by autoclave at 110° C. for ten minutes, thereby obtaining acasein enzymolytic product solution. Then, the obtained enzymolyticproduct solution was dried by spray drying to prepare a powder.

Components contained in the obtained powder were analyzed. Proteins weredetermined by Kjeldahi method and amino acids were measured by an aminoacid analyzer. In addition, a difference after subtracting an amount ofthe amino acids from an amount of the proteins was defined as an amountof peptides. Further, lipids, ash content and water were determined byacid decomposition method, direct ashing method, and drying method byheating under atmospheric pressure, respectively. The remainder aftersubtracting the amount of each component from 100% was defined as anamount of carbohydrates. The results showed that the powder contained35.8% by weight amino acids, 45.7% by weight peptides, 6.6% by weightwater contents, 0.2% by weight lipids, 4.1% by weight ash contents and7.6% by weight carbohydrates.

<Measurement of Amino Acids Composing the Peptide>

The powder prepared above was dissolved in an appropriate amount ofdistilled water and analyzed with an automatic peptide analyzer (tradename PPSQ-10 manufactured by Shimadzu Corporation) to check in whatorder amino acid is located from the N-terminal in the powder. Theautomatic peptide analyzer does not detect any free amino acids.

The total amount of the amino acid located at the 5th residue positionwas 120 pmol and the total amount of the amino acid located at the 6thresidue position was 100 pmol. Based on these results, most peptides inthe above described powder were found to be dipeptides or tripeptides.Additionally, a proportion of the peptides having Pro as the amino acidat the 2nd residue position markedly increased to 49.5%. A proportion ofthe peptides having Pro as the amino acid at the 3rd residue positionwas as high as 29.8%.

Hence the above described powder contains many tripeptides of Xaa ProPro. Thus these peptides were presumably a peptide highly resistant toenzymatic degradation actions of proteases in living bodies.

<Measurement of Peptides Contained in Enzymolytic Products>

For the above described powder of the enzymolytic product, amounts ofthe tripeptides shown in Table 1 contained in the powder was determined,in accordance with a conventional method, using various chemicallysynthesized standard peptides. Results are shown in Table 1.

TABLE 1 Amount of tripeptide (μg/ml) Peptide sequence in 10 mg/ml ofpowder Ser Pro Pro 2.9 Val Pro Pro 29.5 Ile Pro Pro 28.1 Phe Pro Pro27.2 Other Xaa Pro Pro 28.8

The amount of peptides and free amino acids in a solution in which theabove described powder was dissolved and diluted in distilled water was8.15 mg/ml, and an amount of the peptides was 4.57 mg/ml, an amount ofXaa Pro in the peptides was 514.5 μg. The proportion of Xaa Pro based ona total amount of the peptides and free amino acids in the powder wastherefore 6.3% by weight. Further, an amount of Xaa Pro Pro in thepeptide was 116.5 μg and it was thus confirmed a proportion of Xaa ProPro based on a total amount of the peptides and free amino acids in thepowder was 1.4% by weight.

[Example of Preparation of CM4 Fermented Milk Animal Feeds]

Using fermented milk obtained by fermenting a raw material containing amilk protein with the CM4 strain, animal feeds containing the activeingredient Xaa Pro Pro according to the present invention was prepared.

Commercially available powdered non-fat milk was dissolved in distilledwater to the solid content of 9% (w/w) and then autoclaved at 105° C.for ten minutes to sterilize under heating at high temperature. Aftercooled to room temperature, the resulting solution was inoculated with aCM4 strain starter fermentation solution (the number of the bacteria:5×10⁸/ml) at 3% (v/w) and left to stand at 37° C. for 24 hours to yieldCM4 fermented milk.

The obtained CM4 fermented milk was sterilized at a reaching temperatureof 80° C. and then freeze-dried to yield the powder. The obtained freezedried powder was mixed with commercially available powdered feeds (tradename “CE-2”, manufactured by CLEA Japan, Inc.) at the mass ratio of10:90. The mixture was formed into solid feeds to obtain CM4 fermentedmilk feeds. The feeds contained 34.1 mg/kg of Val Pro Pro and 17.1 mg/kgof Ile Pro Pro, both of which peptides were originated from the CM4fermented milk.

[Test 1: Effect of Val Pro Pro (VPP) to Suppress Left Ventricle WallThickening]

The tripeptide Val Pro Pro (VPP) was tested for the effect ofsuppressing the thickening of the left ventricle. The test was carriedout using three groups of male Wistar rats of seven weeks old (JapanSLC, Inc.), each of which group consisted of 9 to 12 rats. Afteracclimation for a week, the animals were received: water with a nitrogenmonoxide synthesis inhibitor, NG-nitro-L-arginine methylesterhydrochloride (L-NAME, manufactured by Sigma) being dissolved at aconcentration of 1 g/L; water with L-NAME and VPP being dissolved at aconcentration of 1 g/L and 0.3 g/L respectively; or water with L-NAMEand angiotensin converting enzyme (AGE) inhibitor, Enalapril at aconcentration of 1 g/L and 0.5 mg/L respectively, was supplied adlibitum for eight weeks. The dose of Enalapril was determined such thatits activity to inhibit ACE was equivalent to VPP's.

The rats were sacrificed by exsanguination under diethyletheranesthesia. The heart was enucleated and then fixed with 10% neutralbuffered formalin solution. Annularly-shaped tissue samples wereprepared from the fixed heart by horizontally excising the lower part ofthe atria such that the right ventricle, septum, left ventricle, andcoronary artery were included. The specimen were embedded in paraffinand cut into thin sections with a thickness of 3.0 to 3.5 μm using amicrotome. Two to five sections per rat were prepared. The sliced thinsections were stained with hematoxylin and eosin and the thickness ofthe left ventricle wall was measured. As for the measurement of thethickness of the ventricle wall, the thickness of a plural of the pointsper one section, each of which point was considered to have an averagethickness, was actually measured with an ocular micrometer under amicroscope. The average thickness was defined as the measured value ofthe section and subjected to statistical analysis. The measurement andevaluation of the thickness was carried out by a pathologist who was notinformed of the name of each sample.

The obtained results are shown in FIG. 1. Compared with the group ofrats fed with L-NAME alone, the group of rats fed with L-NAME and VPPshowed a decrease in the thickness of the left ventricle wall,indicating that VPP suppressed the thickening of the left ventricle walland is thus effective in the prophylaxis of heart failure. In addition,since Enalapril of which dose was set so as to have an equivalent ACEinhibitory activity to VPP did not exhibit the effect, it was confirmedthat the effect did not depend on the ACE inhibition.

[Test 2: Effect of Val Pro Pro (VPP) to Suppress Right Ventricle WallThickening]

In order to check the effect of the tripeptide Val Pro Pro (VPP) tosuppress the thickening of the right ventricle wall as well, a test wascarried out in the same manner as Test 1 and measurement and evaluationwere made. The obtained results are shown in FIG. 2. Compared with thegroup fed with L-NAME alone, the group fed with L-NAME and VPP showed adecrease in the thickness of the right ventricle wall. Hence, VPP alsosuppressed the thickening of the right ventricle wall and was proven tobe effective in the prophylaxis of heart failure. Like in Test 1,Enalapril herein did not show such an effect, it was confirmed that theeffect did not depend on the ACE inhibition.

[Test3: Effect of Ile Pro Pro (IPP) to Suppress Left Ventricle WallThickening]

The tripeptide Ile Pro Pro (IPP) was also tested for the suppressiveeffect against the thickening of the ventricle wall. The test wascarried out using three groups of male Wistar rats of seven weeks old(Japan SLC, Inc.), each of which group consist of 9 to 11 rats. Afteracclimation for a week, the animals received: water with L-NAME(manufactured by Sigma) being dissolved at a concentration of 1 g/L;water with L-NAME and IPP being dissolved at a concentration of 1 g/Land 0.3 g/L respectively; or water with L-NAME and angiotensinconverting enzyme (ACE) inhibitor, Enalapril at a concentration of 1 g/Land 0.5 mg/L respectively, ad libitum for eight weeks. The dose ofEnalapril was determined such that its activity to inhibit ACE wasequivalent to the activity of IPP to inhibit ACE.

The rats were sacrificed by exsanguination under diethyletheranesthesia. The heart was enucleated and then fixed with 10% neutralbuffered formalin solution. Annularly-shaped tissue samples wereprepared from the fixed heart by horizontally excising the lower part ofthe atria such that the right ventricle, septum, left ventricle, andcoronary artery were included. The specimen were embedded in paraffinand cut into thin sections with a thickness of 3.0 to 3.5 μm using amicrotome. Two to five sections per rat were prepared. The sliced thinsections were stained with hematoxylin and eosin and the thickness ofthe left ventricle wall was measured. As for the measurement of thethickness of the ventricle wall, the thickness of a plural of the pointsfor one section, each of which point was considered to have an averagethickness, was actually measured with an ocular micrometer under amicroscope. The average thickness was defined as the measured value ofthe section and subjected to statistical analysis. The measurement andevaluation of the thickness was carried out by a pathologist who was notinformed of the name of each sample.

The obtained results are shown in FIG. 3. Compared with the group ofrats fed with L-NAME alone, the group of rats fed with L-NAME and IPPshowed a decrease in the thickness of the left ventricle wall,indicating that IPP suppressed the thickening of the left ventricle walland is thus effective in the prophylaxis of heart failure. In addition,since Enalapril, of which dose was set so as to have an equivalentactivity to IPP in terms of ACE inhibition, did not exhibit such aneffect, it was confirmed that the effect did not depend on the ACEinhibition.

[Test 4: Effect of Ile Pro Pro (IPP) to Suppress Right Ventricle WallThickening]

In order also to check the effect of the tripeptide Ile Pro Pro (IPP) tosuppress the thickening of the right ventricle wall, a test was carriedout in the same manner as in Test 3, and measurement and evaluation weremade.

The test was carried out using three groups of male Wistar rats of sevenweeks old (Japan SLC, Inc.), each of which group consist of 9 to 12rats. After acclimation for a week, the animal received: water withL-NAME (manufactured by Sigma) being dissolved at a concentration of 1g/L; water with L-NAME and IPP being dissolved at a concentration of 1g/L and 0.3 g/L respectively; or water with L-NAME and angiotensinconverting enzyme (ACE) inhibitor, Enalapril at a concentration of 1 g/Land 0.5 mg/L respectively, ad libitum for eight weeks. The dose ofEnalapril was determined such that its activity to inhibit ACE wasequivalent to the activity of IPP to inhibit ACE.

The rats were sacrificed by exsanguination under diethyletheranesthesia. The heart was enucleated and then fixed with 10% neutralbuffered formalin solution. Annularly-shaped tissue samples wereprepared from the fixed heart by horizontally excising the lower part ofthe atria such that the right ventricle, septum, left ventricle, andcoronary artery were included. The specimen were embedded in paraffinand cut into thin sections with a thickness of 3.0 to 3.5 μm using amicrotome. Two to five sections per rat were prepared. Slices of thethin sections were stained with hematoxylin and eosin and the thicknessof the right ventricle wall was measured. As for the measurement of thethickness of the ventricle wall, the thickness of a plural of the pointsfor one section, each of which point was considered to have an averagethickness, was actually measured with an ocular micrometer under amicroscope. The average thickness was defined as the measured value ofthe section and the thickness of the ventricle wall of each individualwas determined. The measurement and evaluation of the thickness wascarried out by a pathologist who was not informed of the name of eachsample.

The evaluation of the thickening of the ventricle wall involved themeasurement of the thickness of the ventricle wall of six rats of thesame age in weeks, which rats were fed with general tap water with noL-NAME being added. The average of those was defined as a referencevalue. In cases where the thickness of its ventricle wall was largerthan the reference value, a rat was defined as one with the thickening,whereas in cases where the thickness of its ventricle wall was equal toor smaller than the reference value, a rat was considered to be normal.Using the number of the rats with the thickening and the normal rats,comparisons by likelihood-ratio test were made. The results were shownin Table 2 below.

TABLE 2 Evaluation results of the thickening of rat right ventricle wallAdministrated group Thickening Positive Normal L-NAME 5 rats 6 ratsL-NAME and 0 rats 9 rats IPP (**) L-NAME and 1 rat  8 rats Enalapril(**) p < 0.01

The obtained results revealed that IPP also suppressed the thickening ofthe right ventricle wall and thus IPP is also effective on theprophylaxis of heart failure. In addition, since Enalapril, of whichdose was set so as to have an equivalent activity to IPP in terms of ACEinhibition, did not exhibit such an effect, it was confirmed that theeffect did not depend on the ACE inhibition.

[Test 5: Effect of Ile Pro Pro (IPP) to Suppress Heart Septal WallThickening]

In order to check the effect of the tripeptide Ile Pro Pro (IPP) tosuppress the thickening of the heart septal wall as well, a test wascarried out in the same manner as Test 3 and measurement and evaluationwere made.

The obtained results are shown in FIG. 4. Compared with the group fedwith L-NAME alone, the group fed with L-NAME and IPP showed a decreasein the thickness of the heart septal wall, thus indicating that IPPsuppressed the thickening of the heart septal wall and was proven to beeffective in the prophylaxis of heart failure. In addition, sinceEnalapril, of which dose was set so as to have an equivalent ACEinhibitory activity to IPP, did not exhibit the effect, it was confirmedthat the effect did not depend on the ACE inhibition.

Since the agent for suppressing heart wall thickening according to thepresent invention shows effectiveness, even when angiotensin convertingenzyme (ACE) inhibitors do not exhibit the suppressive action, the agentis of great use as a pharmaceutical for the prophylaxis and/or treatmentof cardiac hypertrophy and heart failure, in particular as aprophylactic agent, which agent is not dependent on the ACE inhibitoryactivity. In addition, the active ingredient is a naturally occurringtripeptide originated from foods and the like, it is expected that thepharmaceutical with high efficacy and with little concern for sideeffects is provided. Furthermore, by using or adding the agent forsuppressing heart wall thickening according to the present invention infoods or beverages, the functional foods expected to have theprophylactic effect for cardiac hypertrophy and heart failure areprovided.

REFERENCE

-   1. Japanese Patent No. 2782142-   2. Am J. Hypertens. 1997 August; 10(8):913˜20-   3. J. Clin. Invest. 77, 1993-2000, 1986-   4. Saishin Igaku 48:1404˜1409, 1993-   5. J. Dairy Sci. 1995, 78:777-783-   6. J. Dairy Sci. 1995, 78:1253-1257-   7. Am. J. Clin. Nutr. 1996, 64:767-771

1. An agent for suppressing heart wall thickening, comprising Xaa ProPro as an active ingredient.
 2. The agent for suppressing heart wallthickening according to claim 1, wherein said Xaa Pro Pro is Val Pro Proand/or Ile Pro Pro.
 3. The agent for suppressing heart wall thickeningaccording to claim 1, wherein said Xaa Pro Pro is derived from an animalmilk casein hydrolysate or a concentrate thereof.
 4. The agent forsuppressing heart wall thickening according to claim 3, wherein saidanimal milk casein hydrolysate is a fermented product obtained byfermenting said animal milk casein with Aspergillus.
 5. The agent forsuppressing heart wall thickening according to claim 3 wherein saidanimal milk casein hydrolysate is a hydrolyzed product obtained bydegrading said animal milk casein with an enzyme derived fromAspergillus.
 6. The agent for suppressing heart wall thickeningaccording to claim 5, wherein said enzyme derived from said Aspergillusis an enzyme derived from Aspergillus oryzae.
 7. The agent forsuppressing heart wall thickening according to claim 1, wherein said XaaPro Pro is a fermented product obtained by fermenting a raw materialcontaining a milk protein with a bacterium belonging to the speciesLactobacillus helveticus.
 8. The agent for suppressing heart wallthickening according to claim 7, wherein said bacterium belonging to thespecies Lactobacillus helveticus is Lactobacillus helveticus CM4 strain(FERM BP-6060).
 9. A prophylactic agent for cardiac hypertrophycomprising said agent for suppressing heart wall thickening according toclaim 1 as an active ingredient.
 10. A prophylactic agent for heartfailure comprising said agent for suppressing heart wall thickeningaccording to claim 1 as an active ingredient.
 11. A functional foodcomprising said agent for suppressing heart wall thickening according toclaim
 1. 12. The functional food according to claim 11, which indicatesefficacy for prophylaxis of heart failure.
 13. The agent for suppressingheart wall thickening according to claim 2, wherein said Xaa Pro Pro isderived from an animal milk casein hydrolysate or a concentrate thereof.14. The agent for suppressing heart wall thickening according to claim2, wherein said Xaa Pro Pro is a fermented product obtained byfermenting a raw material containing a milk protein with a bacteriumbelonging to the species Lactobacillus helveticus.
 15. A prophylacticagent for cardiac hypertrophy comprising said agent for suppressingheart wall thickening according to claim 2 as an active ingredient. 16.A prophylactic agent for cardiac hypertrophy comprising said agent forsuppressing heart wall thickening according to claim 3 as an activeingredient.
 17. A prophylactic agent for cardiac hypertrophy comprisingsaid agent for suppressing heart wall thickening according to claim 4 asan active ingredient.
 18. A prophylactic agent for cardiac hypertrophycomprising said agent for suppressing heart wall thickening according toclaim 5 as an active ingredient.
 19. A prophylactic agent for cardiachypertrophy comprising said agent for suppressing heart wall thickeningaccording to claim 6 as an active ingredient.
 20. A prophylactic agentfor cardiac hypertrophy comprising said agent for suppressing heart wallthickening according to claim 7 as an active ingredient.